Although only a small fraction of animal genomes actually code for proteins, recent work has shown that the majority of the genome is still transcribed from DNA into RNA, with much of the later being of unknown function. A portion of these non-coding regions generate short RNA fragments (micro or miRNA and piwi- associated or piRNA) that appear to play a previously-underappreciated and ubiquitous role in gene transcript silencing. Over the last few years, these miRNAs have been implicated in more than 130 different human diseases including numerous forms of cancer. Despite their critical role in cellular function, we know virtually nothing about natural variation in these short regulatory RNAs and how this might influence variation in their mRNA targets. Here we propose to capitalize on the fundamental discoveries regarding miRNA function that have been made using the model nematode Caenorhabditis elegans by performing a comprehensive genomic analysis of small RNA variation and function within the closely related species C. remanei, which is much better suited for studies of natural variation. We aim (1) to determine the evolutionary forces responsible for the origin and divergence of miRNA function and to test the relationship of these evolutionary patterns with the functional properties of the miRNAs;(2) to determine the influence of miRNA regulation on the evolution of mRNA;and (3) to directly test the relationship between miRNA evolutionary and functional divergence. We will accomplish these aims by using next generation technology to sequence the genomes of 32 C. remanei lines drawn from two different populations and 3 additional lines from a recently discovered closely-related incipient species, and by analyzing this variation using established methods from molecular population genetics in addition to a novel "SNP footprinting" approach. We will also use miRNA-system immuno pull-downs to comprehensively identify mRNA targets of the miRNAs. Finally, we use miRNA deletion lines to test the functional consequences of miRNA natural variation. In addition to addressing specific scientific hypotheses, this project will develop a set of genetic and genomic resources that will be uniquely valuable to the broader scientific community. This research uses a natural systems genetics approach that integrates an analysis of micro-evolutionary process with our emerging understanding of regulatory network structure and function within the cell to generate a genome-wide view-dissected with fine- scale SNP variation-of individual transcriptional regulators that is not possible in other major model systems. PUBLIC HEALTH RELEVANCE: Disruption of gene regulation is a major source of human disease, particularly in diseases caused by a breakdown in normal cellular function, such as cancer. Recent studies have made the surprising discovery that a previously unknown class non-coding genetic elements known as micro RNAs play an important role in regulating gene activity by silencing a very large number of genetic targets within the cell. We know virtually nothing about how these regulatory elements vary among individuals within a population. This project uses whole genome sequencing approaches to analyze the forces that determine levels of genetic variation for small regulatory RNAs, such as micro RNAs, within populations and uses functional analyses to examine the role that this variation might have in generating differences in gene regulation among individuals.